Membrane proteins that transport hydrophobic compounds play important roles in multi-drug resistance1–3 and can cause a number of diseases4,5, underscoring the importance of protein-mediated transport of hydrophobic compounds. Hydrophobic compounds readily partition into regular membrane lipid bilayers6, and their transport through an aqueous protein channel is energetically unfavourable3. Alternative transport models, involving acquisition from the lipid bilayer by lateral diffusion have been proposed for hydrophobic substrates3,4,7–12. To date, all transport proteins for which a lateral diffusion mechanism has been proposed function as efflux pumps. Here we present the first example of a lateral diffusion mechanism for the uptake of hydrophobic substrates, by the Escherichia coli outer membrane long-chain fatty acid (LCFA) transporter FadL. A FadL mutant in which a lateral opening in the barrel wall is constricted, but which is otherwise structurally identical to wild-type FadL, does not transport substrates. A crystal structure of FadL from Pseudomonas aeruginosa shows that the opening in the wall of the β-barrel is conserved and delineates a long, hydrophobic tunnel that could mediate substrate passage from the extracellular environment, through the polar lipopolysaccharide layer and, via the lateral opening in the barrel wall, into the lipid bilayer from where the substrate can diffuse into the periplasm. Since FadL homologues are found in pathogenic and biodegrading bacteria, our results have implications for combating bacterial infections and bioremediating xenobiotics in the environment.
Bacterial biodegradation of hydrocarbons, an important process for environmental remediation, requires the passage of hydrophobic substrates across the cell membrane. Here, we report crystal structures of two outer membrane proteins, Pseudomonas putida TodX and Ralstonia pickettii TbuX, which have been implicated in hydrocarbon transport and are part of a subfamily of the FadL fatty acid transporter family. The structures of TodX and TbuX show significant differences with those previously determined for Escherichia coli FadL, which may provide an explanation for the substrate-specific transport of TodX and TbuX observed with in vivo transport assays. The TodX and TbuX structures revealed 14-stranded -barrels with an N-terminal hatch domain blocking the barrel interior. A hydrophobic channel with bound detergent molecules extends from the extracellular surface and is contiguous with a passageway through the hatch domain, lined by both hydrophobic and polar or charged residues. The TodX and TbuX structures support a mechanism for transport of hydrophobic substrates from the extracellular environment to the periplasm via a channel through the hatch domain.membrane protein ͉ x-ray structure
The hydrocarbon-degrading environmental isolate Pseudomonas fluorescens LP6a possesses an active efflux mechanism for the polycyclic aromatic hydrocarbons phenanthrene, anthracene, and fluoranthene but not for naphthalene or toluene. PCR was used to detect efflux pump genes belonging to the resistance-nodulation-cell division (RND) superfamily in a plasmid-cured derivative, P. fluorescens cLP6a, which is unable to metabolize hydrocarbons. One RND pump, whose gene was identified in P. fluorescens cLP6a and was designated emhB, showed homology to the multidrug and solvent efflux pumps in Pseudomonas aeruginosa and Pseudomonas putida. The emhB gene is located in a gene cluster with the emhA and emhC genes, which encode the membrane fusion protein and outer membrane protein components of the efflux system, respectively. Disruption of emhB by insertion of an antibiotic resistance cassette demonstrated that the corresponding gene product was responsible for the efflux of polycyclic aromatic hydrocarbons. The emhB gene disruption did not affect the resistance of P. fluorescens cLP6a to tetracycline, erythromycin, trimethoprim, or streptomycin, but it did decrease resistance to chloramphenicol and nalidixic acid, indicating that the EmhABC system also functions in the efflux of these compounds and has an unusual selectivity. Phenanthrene efflux was observed in P. aeruginosa, P. putida, and Burkholderia cepacia but not in Azotobacter vinelandii. Polycyclic aromatic hydrocarbons represent a new class of nontoxic, highly hydrophobic compounds that are substrates of RND efflux systems, and the EmhABC system in P. fluorescens cLP6a has a narrow substrate range for these hydrocarbons and certain antibiotics.Efflux pumps are prevalent in gram-negative bacteria, in which they contribute to antibiotic resistance and organic solvent tolerance. In pseudomonads, the major efflux pumps belong to the resistance-nodulation-cell division (RND) permease superfamily (22) found in the Bacteria, Archaea, and Eucarya (29). The RND protein, a secondary transporter located in the inner membrane, forms a complex with a membrane fusion protein in the periplasm and an outer membrane channel to effect transport from the cell directly to the extracellular medium (33). Several RND efflux systems, including MexAB-OprM, MexCD-OprJ, MexEF-OprN, and MexXYOprM, have been characterized functionally in Pseudomonas aeruginosa, in which they are involved in the transport of and resistance to antibiotics, hydrophobic dyes, and detergents (22).Recently, similar efflux systems were discovered in bacteria capable of growing in the presence of high concentrations of toxic organic solvents. Both Pseudomonas putida S12 and P. putida DOT-T1E tolerate high concentrations of toluene because they possess RND efflux systems that remove toluene from the cell (12,19,23,24). Toluene and other organic solvents that have octanol-water partition coefficients (log K ow ) between 1.5 and 3.5 accumulate in cell membranes, where they increase membrane permeability, disrupt the m...
Ligand-gated channels, in which a substrate transport pathway is formed as a result of the binding of a small-molecule chemical messenger, constitute a diverse class of membrane proteins with important functions in prokaryotic and eukaryotic organisms. Despite their widespread nature, no ligand-gated channels have yet been found within the outer membrane (OM) of Gram-negative bacteria. Here we show, using in vivo transport assays, intrinsic tryptophan fluorescence and X-ray crystallography, that highaffinity (submicromolar) substrate binding to the OM long-chain fatty acid transporter FadL from Escherichia coli causes conformational changes in the N terminus that open up a channel for substrate diffusion. The OM long-chain fatty acid transporter FadL from E. coli is a unique paradigm for OM diffusion-driven transport, in which ligand gating within a β-barrel membrane protein is a prerequisite for channel formation.
The EmhABC efflux system in Pseudomonas fluorescens cLP6a is homologous to the multidrug and solvent efflux systems belonging to the resistance-nodulation-division (RND) family and is responsible for polycyclic aromatic hydrocarbon transport, antibiotic resistance, and toluene efflux. To gain a better understanding of substrate transport in RND efflux pumps, the EmhB pump was subjected to mutational analysis. Mutagenesis of amino acids within the central cavity of the predicted three-dimensional structure of EmhB showed selective activity towards antibiotic substrates. An A384P/A385Y double mutant showed increased susceptibility toward rhodamine 6G compared to the wild type, and F386A and N99A single mutants showed increased susceptibility to dequalinium compared to the wild type. As well, the carboxylic acid side chain of D101, located in the central cavity region, was found to be essential for polycyclic aromatic hydrocarbon transport and resistance to all antibiotic substrates of EmhB. Phenylalanine residues located within the periplasmic pore domain were also targeted for mutagenesis, and the F325A and F281A mutations significantly impaired efflux activity for all EmhB substrates. One mutation (A206S) in the outer membrane protein docking domain increased antibiotic resistance and toluene tolerance, demonstrating the important role of this domain in transport activity. These data demonstrate the roles of the central cavity and periplasmic domains in the function of the RND efflux pump EmhB.Efflux pumps belonging to the resistance-nodulation-division (RND) family are prevalent in gram-negative bacteria, where they contribute to multidrug and solvent resistance (21, 23). The best-studied RND efflux pumps are AcrB in Escherichia coli and MexB in Pseudomonas aeruginosa, both of which pump a wide variety of structurally diverse compounds, including tetracycline, chloramphenicol, fluoroquinolones, and -lactam antibiotics (20). One of the central questions concerning RND efflux pumps, which recognize and expel a wide variety of structurally diverse hydrophobic compounds, is the mechanism by which these proteins achieve their broad substrate specificity.RND efflux pumps are integral membrane proteins characterized by a large periplasmic domain that is believed to interact with a periplasmic membrane fusion protein and an outer membrane factor protein, forming a three-component efflux system that efficiently expels substrates directly to the extracellular medium (20). The recently determined structure of the E. coli pump AcrB suggested an additional role for the periplasmic domain in substrate recognition and translocation (19). For the trimeric AcrB functional unit, Murakami et al. (19) proposed that substrates enter the pump through vestibules which extend from the external surface of the periplasmic domain and lead into a central cavity located between the transmembrane and periplasmic domains. Each monomer contributes an ␣-helix to form a central pore connecting the central cavity to the upper, funnel-shaped r...
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